Method for the purification of decitabine

ABSTRACT

A method of preparing purified decitabine comprises mixing crude decitabine with solvent, such as dimethylacetamide, to form a solution or suspension and forming the purified decitabine from the solution or suspension. The forming step comprises adding an anti-solvent, such as ethanol, to the solution or suspension. The forming step may further comprise after adding ethanol to provide a mixture of dimethylacetamide and ethanol: cooling the mixture; isolating the solid decitabine present in the cooled mixture; and evaporating residual dimethylacetamide and ethanol from the solid decitabine to provide the purified decitabine. The mixture of dimethylacetamide and ethanol may be heated. The purification method preferably results in decitabine having a ratio of the β-anomer of decitabine to the α-anomer of decitabine of at least about 99.9:0.1.

This application claims the benefit of U.S. Patent Application No.61/708,289 filed Oct. 1, 2012, the disclosure of which is incorporatedherein by reference in it's entirety for all purposes.

FIELD OF THE INVENTION

This invention pertains to the purification of decitabine.

BACKGROUND OF THE INVENTION

The drug compound having the adopted name “decitabine” has chemicalnames:4-amino-1-(2-deoxy-β-D-erythropentofuranosyl)-1,3,5-triazin-2-(1H)-one;or 5-aza-2′-deoxy cytidine; and is structurally represented by formula(I).

Decitabine, a pyrimidine nucleoside analog of cytidine, is used fortreating patients with myelodysplastic syndromes (MDS) includingpreviously treated and untreated, de novo and secondary MDS of allFrench-American-British subtypes (refractory anemia, refractory anemiawith ringed sideroblasts, refractory anemia with excess blasts,refractory anemia with excess blasts in transformation, and chronicmyelomonocytic leukemia) and intermediate-1, intermediate-2, andhigh-risk International Prognostic Scoring System groups.

Decitabine is the active ingredient in the commercially marketed productDACOGEN®, in the form of a sterile lyophilized powder for injection.

U.S. Patent Application Publication No. 2006/0014949 disclosespharmaceutical compositions and methods for treatment of neoplasticconditions using polymorphs of decitabine. This publication alsodiscloses methods for manufacturing and administering suchpharmaceutical compositions. This publication also discloses that twoanomeric forms of decitabine can be distinguished, wherein the β-anomeris the active form of decitabine. The publication mentions various modesof decomposition of decitabine in aqueous solution, namely: (a)conversion of the active β-anomer to the inactive α-anomer; (b) ringcleavage of the aza-pyrimidine ring to formN-(formylamidino)-N′-beta-D-2′-deoxy-(ribofuranosy)-urea; and (c)subsequent forming of guanidine compounds.

U.S. Patent Application Publication No. 2010/0087637 discloses a methodfor producing a β-enriched protected decitabine comprising: a) couplinga protected 2-deoxy-ribofuranose with a protected 5-azacytosine in thepresence of a catalyst to form a reaction mixture comprising a protecteddecitabine; and b) quenching the reaction mixture of step a) with abase. The publication discloses that the ratio of the undesired α-anomerto the desired β-anomer of the protected decitabine precursor is dynamicboth under the reaction conditions of the anomers formed and also duringthe work-up process, specifically due to epimerisation of thecarbohydrate C1 chiral center formed in the coupling reaction. Theundesired α-anomer was found to become enriched following its formationby this epimerisation. The α-anomer was found to be thethermodynamically favored isomer. This publication discloses a method toavoid the undesired enrichment of the α-anomer, to maintain the relativeand absolute amounts of the β-anomer initially formed in the couplingreaction.

U.S. Patent Application Publication No. 2012/0046457 discloses processesfor the preparation and purification of decitabine and processes for thepreparation of a crystalline form of decitabine. The publicationdiscloses a process for purifying decitabine comprising first providinga solution of decitabine in dimethylsulphoxide and then crystallizing asolid from the solution of decitabine and dimethylsulphoxide.Crystallizing can be achieved by combining an anti-solvent with thesolution. Such anti-solvents include an alcohol, an ester, or anymixture thereof, and the anti-solvent in one embodiment is a mixture ofmethanol and ethyl acetate.

SUMMARY OF THE INVENTION

It is highly desirable to provide decitabine which is substantially pureand, in particular, has a high ratio of the β-anomer to the α-anomer ofdecitabine. In fact, according to a guideline issued by theInternational Conference on Harmonization (ICH), the weight ratio of theβ-anomer to the α-anomer of decitabine (“the beta-to-alpha anomerratio”) must be greater than 99.85:0.15. Accordingly, there is a need toprovide a method for purifying crude decitabine and, in particular, toprovide decitabine with a beta-to-alpha anomer ratio of above99.85:0.15, and more preferably above 99.9:0.1.

In view of these and other purposes, the present invention is directedto a method of purifying crude decitabine and the decitabine madethereby. The method of preparing purified decitabine comprises:

-   -   a. mixing crude decitabine with dimethylacetamide to form a        solution or suspension; and;    -   b. forming the purified decitabine as a solid product from the        solution or suspension of step a).

In an embodiment, the step of forming the purified decitabine from thesolution or suspension comprises adding an anti-solvent, such asethanol, to the solution or suspension. The forming step may furthercomprise, after adding ethanol to provide a mixture of dimethylacetamideand ethanol: cooling the mixture of dimethylacetamide and ethanol toprovide a cooled mixture; isolating the solid decitabine present in thecooled mixture; and evaporating residual dimethylacetamide and ethanolfrom the solid decitabine to provide the purified decitabine.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary, but are notrestrictive, of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Several considerations were taken into account in developing a methodfor purifying decitabine. First, decitabine is not soluble in manysolvents. As mentioned in the '949 Publication, decitabine is generallypoorly soluble in many solvents. A notable exception is methylsulfoxide, in which the compound was found be soluble to the extent ofapproximately 37 mg/mL. Decitabine is also slightly soluble in1,1,1,3,3,3-hexafluoro-2-propanol (about 18 mg/mL) and sparingly solublein water (about 8 mg/mL). Moreover, it was found that it was undesirableto use large volumes of solvent for reasons of expense and processcapacity. Furthermore, it was found that the inclusion of water in theprocess, especially at high temperatures, causes degradation ofdecitabine, particularly by causing hydrolysis. As stated in the '637published application, decitabine exists as the α-anomer and theβ-anomer, and the β-anomer is desired but the α-anomer is thethermodynamically favored isomer.

With all of this in mind, a process for purifying decitabine wasdeveloped. The process comprises: a) mixing crude decitabine with asolvent, such as dimethylacetamide, to form a solution or suspension;and b) forming the purified decitabine as a solid product from thesolution or suspension of step a). In an embodiment, the step of formingthe purified decitabine from the solution or suspension comprises addingan anti-solvent, such as ethanol, to the solution or suspension. Theforming step may further comprise, after adding ethanol to provide amixture of dimethylacetamide and ethanol: cooling the mixture ofdimethylacetamide and ethanol to provide a cooled mixture; isolating thesolid decitabine present in the cooled mixture; and evaporating residualdimethylacetamide and ethanol from the solid decitabine to provide thepurified decitabine. The purification method results in a purifieddecitabine product having a beta-to-alpha anomer ratio of at least99.9:0.1 when the beta-to-alpha anomer ratio of the crude decitabine ispreferably between about 90:10 to about 99:1, and more preferablybetween about 95:5 to about 99:1. The purification method of theinvention improves the beta-to-alpha anomer ratio regardless of thebeta-to-alpha anomer ratio of the crude decitabine, but is especiallyadvantageous when the beta-to-alpha anomer ratio of the crude decitabineis less than about 99.5:0.5 and preferably less than about 99:1. As usedherein, the purity values are in terms of area under a chromatographiccurve (AUC) and purity was measured by HPLC assay.

In carrying out the method for preparing purified decitabine, firstcrude decitabine is mixed with a solvent, such as dimethylacetamide, toform a solution or a suspension. The solvent selected can be any knownsuitable solvent having an adequate solubility for decitabine.Preferably, decitabine is at least moderately soluble in the solventused to avoid using too much solvent. It has been found that preferablyaprotic amide solvents are used, such as dimethylacetamide, dimethylformamide (DMF), and/or N-methylpyrrolidinone (NMP).

The crude decitabine used in this step may be from any source.Typically, such crude decitabine has a beta-to-alpha anomer ratio ofbetween about 99:1 and 95:5 and more typically about 99:1. Exemplarymethods for synthesizing decitabine may be found in U.S. PatentPublication Nos. 2010/0087637 and 2012/0046457.

In one embodiment, crude decitabine is partially dissolved in a solvent,such as dimethylacetamide, at room temperature and the mixture is heateduntil complete dissolution of the decitabine or to a temperature abovethe point at which the decitabine is fully dissolved. Certainnon-decitabine components or impurities may remain undissolved. In oneembodiment, crude decitabine from any suitable synthesis process, suchas those described above, is first suspended in dimethylacetamide toform a mixture. Relative amounts of decitabine and dimethylacetamide maybe used such that the amount of decitabine is above the solubility limitat the temperature in which the mixture is formed. Then, the mixture maybe heated to a temperature such that the decitabine completely dissolvesor to a temperature above that temperature. Preferably, the heating ofthe mixture of the suspension of crude decitabine and dimethylacetamideis to a temperature of from about 55° C. to about 70° C. for a time offrom about 10 to about 30 minutes. The time and temperature should beselected to ensure that the decitabine is preferably fully dissolved.Alternatively, an amount of solvent may be used such that the decitabinefully dissolves in the solvent at the temperature of mixing (e.g., roomtemperature). Then, the solution may be filtered to remove insolubleimpurities, such as non-decitabine components or impurities, and thefiltrate may again be heated and stirred if necessary to ensure that thedecitabine is fully dissolved.

The process next involves forming the purified decitabine from thesolution or suspension produced by the mixing step. This forming stepmay involve adding an anti-solvent to the solution or suspension. Theanti-solvent used can be any suitable anti-solvent known in the art toaid in forming solid decitabine from a mixture of decitabine anddimethylacetamide. Such anti-solvents may include methanol, isopropanol,ethyl acetate, acetone, methyl ethyl ketone, and acetonitrile, butpreferably the anti-solvent is ethanol. The amount of anti-solvent usedis preferably an amount sufficient to form solid decitabine, uponcooling, of at least 50%, or at least 80% of the decitabine added ascrude decitabine. In a system in which ethanol is used as theanti-solvent and dimethylacetamide is used as the solvent, the amount ofethanol added may be about 70% to about 85% v/v, more preferably about75% to about 78% v/v. As used herein, the term “% v/v” refers to thepercent by volume of the identified solvent based on the sum of thepre-mixed individual volumes of all of the solvents and anti-solvents.In an embodiment in which ethanol is used as the anti-solvent anddimethylacetamide is used as the solvent, it has been found that thesolution prior to addition of the ethanol becomes cloudy upon additionof ethanol.

The product formed after adding the anti-solvent may optionally beheated prior to a cooling step, discussed below. It has been found that,in an embodiment in which ethanol is used as the anti-solvent anddimethylacetamide is used as the solvent, the solution remains cloudyduring the heating. In this embodiment of the invention, the heating ofthe product after adding ethanol may be for a time of about 10 to about90 min at a temperature of from about 50° C. to about 75° C., preferablyfor a time of from about 30 to about 60 min at a temperature of fromabout 60° C. to about 70° C.

As mentioned above, the forming step may further comprise, after addingethanol to provide a mixture of dimethylacetamide and ethanol, coolingthe mixture of dimethylacetamide and ethanol to provide a cooled mixtureand to form solid decitabine. Cooling causes solid decitabine to dropout of the solution. It is believed that the α-anomer is more soluble inthis solution than the β-anomer, thereby leading to a solid decitabineproduct which is more pure (i.e., has a higher beta-to-alpha anomerratio). Preferably, the cooling step comprises cooling the product,either directly after the addition of ethanol or after the optionalheating step, to a temperature of from about 0° C. to about 20° C., morepreferably, from about 5° C. to about 15° C.

Thereafter, the forming step may involve isolating solid decitabinepresent in the cooled mixture from the solvent system. This may includefiltering the solid decitabine from the solvent system. Any knownfiltering system known in the art may be used for this purpose, such asBuchner filter funnels or other filtration/collection apparatus.

Next, the final part of the forming step may involve evaporating thesolvent. In particular, any residual solvent is evaporated from theproduct of the isolation step to form the purified decitabine. This may,for example, involve exposing the product to a reduced pressure byapplying vacuum. In addition, the product after the filtering step maybe rinsed with a solvent prior to performing the evaporating step.Preferably, that solvent is ethanol. In sum, the evaporating step isdone to dry any residual solvent from the product and may include dryingfor a period of 24 hours or more, which provides the purified decitabineas a solid product.

Preferably, the entire method is run such that the temperature of theprocess never exceeds 80° C., more preferably not exceeding 70° C. Evenmore preferably, the addition of or exposure to water is avoided inorder to limit the degradation of decitabine by hydrolysis. The methodof the present invention is capable of providing decitabine with abeta-to-alpha anomer ratio of at least 99.9:0.1. In addition, thedecitabine content of the purified product, as purified according to thesteps discussed below in the examples, was at least 99%.

EXAMPLE

The following example is included to more clearly demonstrate theoverall nature of the present invention.

Solid crude decitabine (1.0 g) was partially dissolved indimethylacetamide (10 mL, Sigma-Aldrich, Fluka, 44901-1L) and themixture was then heated in a bath set at 65° C. The decitabine in thesolid crude decitabine was dissolved completely when the bathtemperature reached 38° C., although certain less soluble non-decitabinecomponents and impurities remained in solid form. The solution wasfiltered through a 0.45 μm syringe filter (PTFE) to remove thenon-decitabine components and impurities, and the filter was rinsed withdimethylacetamide (1 mL). The filtrate was heated and stirred at 55° C.,which was the bath temperature. Ethanol (35 mL, 200 proof, 99.5%) atroom temperature was added slowly, while keeping the temperature of themixture between 40-55° C., and the solution became cloudy upon additionof ethanol After the ethanol addition was completed, the mixture washeated to 65° C. and maintained at this temperature for approximately 30min. The mixture was then gradually cooled to room temperature withstirring, then to 7° C.

Solid cake product was filtered in a glove bag under nitrogen. The cakewas washed with anhydrous ethanol (3×3 mL) and dried under vacuum on thefilter in a glove bag for 15 min. Then, the solid was transferred to avial and dried at reduced pressure (about 6 mm Hg) for 18 hours atambient temperature to give 0.81 grams of purified decitabine (81%recovery) as a solid product. The isomeric purity was 99.9% AUC of thedesired β-anomer (i.e., the purified product had a beta-to-alpha anomerratio of 99.9:0.1), and the solid form matched a decitabine standard(from methanol crystallization) by XRPD, having XRPD peaks locatedapproximately at 7.1, 12.9, 14.2, 18.9, 21.3, 23.8, 24.8 and 26.6±0.2°2Θ.

Residual ethanol content by GC with headspace analysis (AgilentTechnologies GC Model #7890 with headspace attachment Model #G1888)after drying under vacuum of around 6 mm Hg at 22° C. for 24 hours was3960 ppm (ICH limit 5000 ppm), and residual dimethylacetamide wasnon-detectable.

Although the invention is illustrated and described herein withreference to specific embodiments, the invention is not intended to belimited to the details shown. Rather, various modifications may be madein the details within the scope and range of equivalents of the claimsand without departing from the invention.

What is claimed:
 1. A method of preparing purified decitabine, comprising: a) mixing crude decitabine with dimethylacetamide to form a solution or suspension; and b) forming the purified decitabine as a solid product from the solution or suspension of step a).
 2. The method of claim 1, wherein step b) comprises adding ethanol to the solution or suspension.
 3. The method of claim 2, wherein step b) further comprises, after adding ethanol to provide a mixture of dimethylacetamide and ethanol: cooling the mixture of dimethylacetamide and ethanol to provide a cooled mixture; isolating solid decitabine present in the cooled mixture; and evaporating residual dimethylacetamide and ethanol from the solid decitabine to provide the purified decitabine.
 4. The method of claim 3, further comprising, between the steps of adding ethanol and cooling, heating the mixture of dimethylacetamide and ethanol.
 5. The method of claim 4, wherein the heating step comprises heating the mixture of dimethylacetamide and ethanol to a temperature of from about 50° C. to about 75° C. and maintaining for a time of about 10 to about 90 min.
 6. The method of claim 4, wherein the heating step comprises heating the mixture of dimethylacetamide and ethanol to a temperature of from about 60° C. to about 70° C. and maintaining for a time of from about 30 to about 60 min.
 7. The method of claim 1, wherein the temperature of the process does not exceed 80° C.
 8. The method of claim 1, wherein the temperature of the process does not exceed 70° C.
 9. The method of claim 1, wherein the mixing step comprises suspending the crude decitabine in dimethylacetamide to form a mixture of crude decitabine and dimethylacetamide and heating the mixture of crude decitabine and dimethylacetamide to a temperature sufficient to dissolve the crude decitabine.
 10. The method of claim 9, wherein heating the mixture of crude decitabine and dimethylacetamide comprises heating to a temperature of from about 55° C. to about 70° C. for a time of from about 10 to about 30 min.
 11. The method of claim 2, wherein the amount of ethanol added comprises from about 70% to about 85% v/v.
 12. The method of claim 2, wherein the amount of ethanol added comprises from about 75% to about 78% v/v.
 13. The method of claim 3, wherein the cooling step comprises cooling the mixture of dimethylacetamide and ethanol to a temperature of from about 0° C. to about 20° C.
 14. The method of claim 1, wherein the cooling step comprises cooling the mixture of dimethylacetamide and ethanol to a temperature of from about 5° C. to about 15° C.
 15. The method of claim 3, wherein isolating the solid decitabine comprises filtering.
 16. The method of claim 3, wherein evaporating comprises applying vacuum to the solid decitabine.
 17. The method of claim 3, further comprising rinsing the solid decitabine with a solvent after the isolating step and before the evaporating step.
 18. The method of claim 17, wherein the solvent is ethanol.
 19. A method of preparing purified decitabine, comprising a) mixing crude decitabine with dimethylacetamide to form a solution or suspension; b) adding ethanol to the solution or suspension in an amount of from about 70% to about 85% v/v; c) cooling the product of step b) to a temperature of between about 5° C. to about 15° C. to provide a cooled mixture; d) isolating solid decitabine present in the cooled mixture; and e) evaporating residual dimethylacetamide and ethanol from the solid decitabine to provide the purified decitabine, wherein the temperature of the process throughout does not exceed 80° C.
 20. The method of claim 19, further comprising, between steps b) and c), heating the mixture of dimethylacetamide and ethanol for a time of between 30 and 60 min and at a temperature of between 60° C. and 70° C.
 21. A crystalline form of decitabine having an X-ray powder diffraction pattern comprising peaks, in terms of 2-theta values, at 7.1, 12.9, 14.2, 18.9, 21.3, 23.8, 24.8 and 26.6 +/−approximately 0.2 made by a process comprising the steps of: a) mixing crude decitabine with dimethylacetamide to form a solution or suspension; and b) forming the decitabine as a solid product from the solution or suspension of step a).
 22. Decitabine having a beta-to-alpha anomer ratio of at least 99.9:0.1 made by a process comprising the steps of: a) mixing crude decitabine with dimethylacetamide to form a solution or suspension; and b) forming the decitabine as a solid product from the solution or suspension of step a). 